Recombinant Invertebrate iridescent virus 3 Uncharacterized protein 033L (IIV3-033L)
Description
Introduction to Recombinant Invertebrate Iridescent Virus 3 Uncharacterized Protein 033L (IIV3-033L)
The Recombinant Invertebrate Iridescent Virus 3 Uncharacterized Protein 033L (IIV3-033L) is a protein derived from the Invertebrate Iridescent Virus 3 (IIV-3), also known as the Mosquito Iridescent Virus. This virus belongs to the genus Chloriridovirus and is part of the family Iridoviridae, which primarily infects invertebrates . The IIV3-033L protein is expressed in Escherichia coli (E. coli) and is fused with an N-terminal His tag for purification purposes .
Characteristics of IIV3-033L Protein
The IIV3-033L protein is a full-length recombinant protein consisting of 194 amino acids. It is provided in a lyophilized powder form and has a purity of greater than 90% as determined by SDS-PAGE. The protein is stored at -20°C or -80°C and should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, adding 5-50% glycerol is recommended .
Table 2: Genome Features of IIV-3
| Feature | Description |
|---|---|
| Genome Size | Approximately 190 kbp |
| GC Content | 48% |
| Number of Genes | 126 predicted genes |
| Unique Genes | 33 genes without homologues in other IVs |
References
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Creative Biomart. Recombinant Full Length Invertebrate Iridescent Virus 3 Uncharacterized Protein 033L(Iiv3-033L) Protein, His-Tagged. [Accessed 2025].
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Creative Biomart. Recombinant Full Length Invertebrate Iridescent Virus 3 Uncharacterized Protein 033L(Iiv3-033L) Protein, His-Tagged. [Accessed 2025].
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PubMed Central. Genome of Invertebrate Iridescent Virus Type 3. [Accessed 2025].
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
Tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: vg:4156343
Q&A
What is Invertebrate iridescent virus 3 (IIV-3) and how does IIV3-033L fit within its genome?
IIV-3, also known as mosquito iridescent virus (MIV), is the type species and currently sole member of the genus Chloriridovirus within the family Iridoviridae. IIV-3 is characterized by its restricted host range (mosquitoes [Diptera]) and relatively large particle size (180 nm) .
The IIV-3 genome is 190,132 bp in length with 48% G+C content, which differs significantly from previous size estimates . The genome contains 126 predicted open reading frames (ORFs) encoding proteins of 60 to 1,377 amino acids in length . IIV3-033L is one of these ORFs, representing an uncharacterized protein within the viral genome.
Key genomic features of IIV-3:
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Approximately 20% of the genome consists of repetitive DNA
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Contains 33 unique genes not found in other iridoviruses
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Shares 27 gene homologues present in all sequenced iridoviruses
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Contains 52 genes present in IIV-6 (Chilo iridescent virus) but absent in vertebrate iridoviruses
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Shows no obvious colinearity with other completely sequenced iridovirus genomes
What expression systems are optimal for producing recombinant IIV3-033L?
Based on the protein characteristics and viral origin, several expression systems could be employed:
| Expression System | Advantages | Disadvantages | Optimization Strategies |
|---|---|---|---|
| Bacterial (E. coli) | High yield, cost-effective, rapid expression | Limited post-translational modifications, potential membrane protein misfolding | Use specialized strains (C41/C43), low induction temperature, fusion partners (SUMO, MBP) |
| Insect cell (Baculovirus) | Native-like environment for insect virus protein, better membrane protein folding | Higher cost, longer production time | Codon optimization, signal sequence modification, cell line selection (Sf9, High Five) |
| Mammalian cell | Most authentic post-translational modifications | Lower yields, highest cost | Transient vs. stable expression optimization, serum-free adaptation |
| Cell-free | Rapid, controllable environment, suitable for toxic proteins | Lower yields | Supplementation with lipids/detergents for membrane proteins |
For membrane-associated proteins like IIV3-033L, insect cell expression using baculovirus vectors would provide a more native-like environment, potentially resulting in proper folding and post-translational modifications relevant to the protein's function in mosquito hosts .
What purification strategies can be employed for recombinant IIV3-033L?
Purification of IIV3-033L requires strategies appropriate for membrane-associated proteins:
| Purification Method | Considerations for IIV3-033L | Technical Notes |
|---|---|---|
| Affinity Chromatography | Primary method using appropriate tag (His, GST, FLAG, etc.) | Tag position (N vs. C-terminal) may affect function; detergent selection critical |
| Detergent Extraction | Essential for membrane proteins | Screen multiple detergents (DDM, LMNG, CHAPS); use lowest effective concentration |
| Size Exclusion Chromatography | Secondary purification step | Separates monomeric protein from aggregates; assess oligomeric state |
| Ion Exchange Chromatography | Based on protein's theoretical pI | Orthogonal to affinity purification; salt gradient optimization |
| Tag Removal | May be required for functional studies | Protease selection (TEV, PreScission); second affinity step |
| Stability Assessment | Critical for downstream applications | Thermal shift assays with different buffers/additives |
According to the product information, commercially available recombinant IIV3-033L is provided with a tag, though the specific tag type "will be determined during production process" . The protein is stored in a Tris-based buffer with 50% glycerol, optimized for stability .
What analytical methods should be used to verify the quality of purified IIV3-033L?
A comprehensive quality assessment would include:
| Analytical Method | Purpose | Expected Results |
|---|---|---|
| SDS-PAGE | Purity assessment, approximate molecular weight | Single band at ~22 kDa (194 aa + tag) |
| Western Blotting | Specificity confirmation | Specific binding of anti-tag or anti-IIV3-033L antibodies |
| Mass Spectrometry | Exact mass determination, sequence verification | Mass matching theoretical value; peptide coverage >80% |
| Circular Dichroism | Secondary structure assessment | Spectrum consistent with predicted α-helical content (from transmembrane region) |
| Size Exclusion Chromatography | Oligomeric state, aggregation assessment | Homogeneous peak at expected elution volume |
| Dynamic Light Scattering | Monodispersity analysis | Single peak with low polydispersity index |
| Thermal Shift Assay | Stability assessment | Melting temperature indicative of stable, folded protein |
| Functional Assays | Verification of biological activity | Dependent on hypothesized function (e.g., membrane binding) |
These methods collectively provide a comprehensive profile of protein quality, essential for ensuring reproducible experimental results in downstream applications.
How can researchers determine the membrane topology of IIV3-033L?
Given the predicted membrane association of IIV3-033L, determining its topology is crucial:
| Approach | Methodology | Expected Outcomes |
|---|---|---|
| Computational Prediction | TMHMM, Phobius, MEMSAT algorithms | Number of transmembrane domains; orientation prediction |
| Protease Protection Assay | Limited proteolysis of membrane-embedded protein | Identification of protected vs. exposed domains |
| Glycosylation Mapping | Introduction of N-glycosylation sites; glycosylation indicates luminal exposure | Map of luminal/cytoplasmic domains |
| Cysteine Accessibility | Chemical modification of engineered cysteine residues | Identification of solvent-accessible regions |
| GFP-fusion Analysis | C/N-terminal GFP fusion fluorescence in different compartments | Determination of C/N terminus orientation |
| Antibody Epitope Mapping | Generation of antibodies to different protein regions; accessibility without permeabilization | Map of exposed epitopes |
| Cryo-EM | Single-particle analysis of protein in nanodisc/liposome | Direct visualization of membrane insertion |
The N-terminal hydrophobic region (WLYLLVFGCLSVLVLVLV) would be the primary focus for topology analysis, as it likely represents a membrane-spanning segment or membrane-association domain .
What approaches can elucidate the function of IIV3-033L in the viral life cycle?
A systematic functional investigation would involve:
| Approach | Methodology | Research Questions Addressed |
|---|---|---|
| Viral Genetics | CRISPR-Cas9 genome editing of viral genome; phenotypic analysis | Is IIV3-033L essential for viral replication? What stage is affected by deletion? |
| Reverse Genetics | Generation of recombinant viruses with tagged or mutated IIV3-033L | Which domains/residues are critical for function? |
| Temporal Expression Analysis | Time-course of protein expression during infection | When is IIV3-033L expressed (early/late)? |
| Subcellular Localization | Immunofluorescence or live-cell imaging with fluorescent tags | Where does IIV3-033L localize during infection? |
| Protein-Protein Interactions | Co-immunoprecipitation, proximity labeling, yeast two-hybrid | What viral or host proteins interact with IIV3-033L? |
| Lipidomics | Analysis of lipid composition changes induced by IIV3-033L | Does IIV3-033L alter membrane structure or composition? |
| Host Range Analysis | Complementation studies in different insect cell lines | Does IIV3-033L contribute to host specificity? |
Given IIV-3's specific tropism for mosquito hosts, particularly affecting the fat body, dermis, and other tissues , these approaches could reveal if IIV3-033L contributes to this tissue specificity or other aspects of the virus-host interaction.
What protein interaction networks might involve IIV3-033L during infection?
Protein interaction studies could reveal IIV3-033L's functional context:
| Interaction Type | Experimental Approach | Potential Biological Significance |
|---|---|---|
| Viral Structural Proteins | Proximity labeling during virion assembly | Role in virion structure/morphogenesis |
| Viral Non-structural Proteins | Immunoprecipitation-mass spectrometry during replication | Involvement in replication complex |
| Host Membrane Proteins | Split-ubiquitin membrane yeast two-hybrid | Role in entry, membrane reorganization |
| Host Immune Factors | CRISPR screening for host factors affecting IIV3-033L function | Immune evasion or manipulation |
| Host Trafficking Machinery | Fluorescence co-localization with cellular markers | Role in viral trafficking or egress |
IIV-3 infects mosquito larvae, with replication primarily in the fat body and to a lesser extent in the dermis, imaginal disks, trachea, gonads, and hemocytes . Protein interaction studies focusing on these tissues could provide particularly relevant insights.
How might post-translational modifications regulate IIV3-033L function?
Analysis of potential post-translational modifications (PTMs):
| PTM Type | Prediction Methods | Validation Approaches | Functional Implications |
|---|---|---|---|
| Phosphorylation | NetPhos, GPS, Scansite | Mass spectrometry, phospho-specific antibodies | Regulation of protein-protein interactions, cellular localization |
| Glycosylation | NetNGlyc, NetOGlyc | PNGase F treatment, lectin blotting | Protein folding, stability, immune evasion |
| Palmitoylation | CSS-Palm, MDD-Palm | Acyl-biotin exchange assay, metabolic labeling | Enhanced membrane association, localization to lipid rafts |
| Ubiquitination | UbPred, UbiSite | Ubiquitin pull-down, mass spectrometry | Protein turnover, regulation of abundance |
| SUMOylation | GPS-SUMO, SUMOplot | SUMO-specific immunoprecipitation | Protein-protein interactions, spatial regulation |
The amino acid sequence of IIV3-033L contains numerous potential modification sites, including multiple serine and threonine residues that could be phosphorylated . Mutations of these sites, followed by functional assays, could reveal their importance in protein regulation.
What structural biology approaches are most suitable for membrane-associated proteins like IIV3-033L?
Obtaining structural information for IIV3-033L requires specialized approaches:
| Method | Advantages | Challenges | Optimization Strategies |
|---|---|---|---|
| X-ray Crystallography | Highest resolution potential | Difficult crystallization of membrane proteins | LCP crystallization, fusion with crystallization chaperones |
| Cryo-Electron Microscopy | No crystallization required; native-like environment | Lower resolution for small proteins | Incorporation into nanodiscs; antibody fragment complexes |
| Nuclear Magnetic Resonance | Dynamic information; solution conditions | Size limitations; extensive isotope labeling | Focus on soluble domains; deuteration |
| Small-Angle X-ray Scattering | Low-resolution envelope in solution | Limited detailed information | Complement with computational modeling |
| Electron Paramagnetic Resonance | Specific distance measurements | Requires spin labeling | Strategic cysteine introduction |
| Hydrogen-Deuterium Exchange MS | Conformational dynamics information | Complex data analysis | Focus on solvent-accessible regions |
| AlphaFold/RoseTTAFold | No experimental protein required | Accuracy varies with template availability | Refinement with experimental constraints |
A hybrid approach combining computational prediction, lower-resolution experimental methods, and focused high-resolution studies of specific domains would likely be most successful for IIV3-033L.
How can researchers develop assays to test hypothesized functions of IIV3-033L?
Based on its characteristics, several functional hypotheses could be tested:
| Hypothesized Function | Assay Design | Expected Results if Hypothesis Correct |
|---|---|---|
| Membrane Fusion/Penetration | Liposome fusion assay with fluorescent lipids | IIV3-033L induces mixing of lipid bilayers |
| Pore Formation | Ion flux measurements in proteoliposomes | Specific ion conductance patterns |
| Replication Complex Formation | Membrane reorganization visualization in cells | Co-localization with viral replication markers |
| Host Protein Interaction | Pull-down with mosquito cell lysates | Specific host protein binding |
| Immune Evasion | Interferon response assays in presence/absence of protein | Suppression of insect immune signaling |
| Virion Assembly | EM localization in virus particles | Specific positioning in viral envelope |
These assays should be performed with appropriate controls, including mutated versions of IIV3-033L where key functional residues are altered.
How does IIV3-033L compare to proteins from other iridoviruses, and what does this reveal about evolution?
Comparative analysis across the Iridoviridae family:
| Analytical Approach | Methodology | Evolutionary Insights |
|---|---|---|
| Sequence Homology | PSI-BLAST, HHpred against all iridovirus genomes | Identification of orthologous proteins; conservation level |
| Phylogenetic Analysis | Maximum likelihood trees of homologous proteins | Evolutionary relationships; selection pressures |
| Synteny Analysis | Genome organization comparison across iridoviruses | Conservation of genomic context |
| Structure Prediction Comparison | AlphaFold models of homologs; structural alignment | Conservation of structural features despite sequence divergence |
| Domain Architecture | InterProScan, HMMER profile comparison | Evolution of domain organization |
| Selection Analysis | dN/dS ratio calculation on aligned sequences | Identification of positively selected sites |
IIV-3 is distantly related to other iridovirus genera, showing low levels of amino acid identity to homologs in other iridoviruses . The IIV-3 genome contains 27 gene homologs present in all sequenced iridoviruses and 52 genes present in IIV-6 but not in vertebrate iridoviruses . Determining whether IIV3-033L belongs to the core conserved genes or is specific to invertebrate iridoviruses would provide insight into its evolutionary significance.
